Abstract: Molecular study of plants relies on high quality and yield of the DNA obtained. The leaf of banana contains high levels of polysaccharides, polyphenols and secondary metabolites. Hence, we developed an easy and efficient protocol for isolation of genomic DNA from leaves of four banana cultivars (Grand Naine, Poovan, Nendran and Red Banana). The method prevents solubilization of polysaccharides and polyphenols using high concentration of salt in combination with polyvinyl pyrrolidone and Cetyl trimethyl ammonium bromide. The purity of DNA was confirmed by 260/280 ratio from spectrophotometer readings and further by restriction digestion of the isolated DNA using Hind III and EcoRI. The yield of DNA ranged from 712-808 μg g-1 and the purity ratio was ranging from 1.7 to 1.8 showing minimal level of secondary metabolite contamination. Thus, this protocol proved amenable for PCR analysis and can be applicable to other plant related species also. Further more this protocol was suitable for randomly amplified polymorphic DNA analysis to study genetic variation in four banana cultivars.